Sweetening compositions and processes for preparing them

ABSTRACT

The invention relates to sweetening compositions containing from about 16% to about 75% mogroside V and from about 30% to about 95% total terpene glycosides on a dry weight basis, and wherein a filtered (0.2 μm) solution of the composition in water having a solids content of 1% w/v has an absorbance at 420 nm of about 0.55 or below. The invention also relates to methods of preparing such compositions.

FIELD OF THE INVENTION

This invention relates to sweetening compositions. It more particularlyrelates to sweetening compositions containing terpene glycosides, and tofoods and beverages sweetened with such compositions. The invention alsorelates to processes for preparing the sweetening compositions.

BACKGROUND OF THE INVENTION

With obesity on the rise in the Western world, consumers are constantlylooking for ways to reduce the calorie content of their diet, butwithout sacrificing flavour. Many lower calorie food and beverageproducts have been developed. There are a number of low calorie productscontaining artificial non-nutritive sweeteners, such as saccharine,aspartame, cyclamate, dipeptides, trichlorosucrose and Acesulfame K.However, there is growing concern over the safety of some of theseartificial sweeteners and many consumers would prefer to reduce theirintake of such sweeteners.

Certain naturally-occurring terpene glycosides, particularly triterpeneglycosides, are both intensely sweet and non-calorific. For thesereasons, triterpene glycosides are very attractive for use as asweetening agent in the food, beverage and dietary industries.

Luo Han Guo refers to the fruit of Siraitia grosvenorii (formerly knownas Momordica grosvenorii), a member of the Cucurbitaceae family. Luo HanGuo is grown in the South East provinces of China, mainly in the Guangxiregion. It has been cultivated and used for hundreds of years as atraditional Chinese medicine to treat coughs and congestion of thelungs, and also as a sweetener and flavouring agent in soups and teas.

Luo Han Guo and other fruit of the Cucurbitaceae family contain terpeneglycosides known as mogrosides and siamenosides, which are present at alevel of around 1% of the fleshy part of the fruit. These compounds havebeen described and characterized by

Matsumoto et al; Chem. Pharm. Bull., 38(7), 2030-2032 (1990). Mogrosidesare compounds in which between one and six glucose molecules areattached to a triterpene backbone. The most abundant mogroside ismogroside V, which has been estimated to have a sweetness ofapproximately 250% of that of cane sugar, on a weight basis.

The Luo Han Guo fruit itself, although sweet, is unsuitable forwidespread use as a non-nutritive sweetener without additionalprocessing. The raw fruit has a tendency to easily form off-flavours byfermentation. Also, its pectin eventually gels. Drying the fruitpreserves it, but also causes the formation of other undesirable bitter,astringent and cooked flavours. A number of methods have been describedfor processing Luo Han Guo to remove undesired flavour components, toproduce extracts containing mogrosides, that have a flavour profile moreacceptable for use as sweetening compositions.

Experientia 31(5) 533-534, 1975 (Lee, C.-H.) describes a process forextracting mogrosides from Luo Han Guo using hot water extraction,followed by passing the extract containing mogrosides through anAmberlite resin that retains the mogrosides, and eluting the mogrosideswith 50% ethanol.

U.S. Pat. No. 5,411,755 describes a process for preparing a sweet juicecontaining mogrosides and sugar from Luo Han Guo fruit. The processinvolves separating peel and seeds from unprocessed juice of the fruit,acidifying the juice, removing off-flavour precursors from the fruit,and removing a methylene chloride extractable volatiles fraction fromthe juice.

U.S. Pat. No. 5,433,965 describes a sweetener composition comprising acombination of sugar and a sweet juice containing mogrosides derivedfrom Luo Han Guo fruit.

U.S. patent application Ser. No. 10/086,322 describes a sweetener blendcomposition comprising Luo Han Guo fruit concentrate, fructose andmaltodextrin.

U.S. Pat. No. 6,124,442 describes a process for preparing a drycomposition containing mogrosides and derived from Luo Han Guo fruit.The process includes obtaining a liquid extract from Luo Han Guo andmixing the extract with a solution saturated with at least one elementhaving an oxidation number of one or two. The resulting mixture providesfor a solid precipitate material and a liquid portion containing thetriterpene glycosides which is then passed through a macroporous resin.The resin is then washed with an alcohol to obtain a solution thereofcontaining the triterpene glycosides. The solution is condensed toprovide a purified liquid triterpene glycoside solution and then adrying step is performed to obtain a dry composition containing thetriterpene glycosides.

United States patent application publication 20060003053A1 describes aprocess for extracting juice from a plant material containing triterpeneglycosides. The process involves the steps of crushing the plantmaterial, blanching the crushed plant material in acidified water toobtain a puree comprising a juice extract and a plant solids residue,separating the juice extract from the plant solids residue, mixing anenzyme with the juice extract and separating the juice extract to obtaina sweet juice.

Chinese patent 1015264 describes a process for preparing a drycomposition containing mogrosides and derived from Luo Han Guo fruit.The process includes obtaining a liquid extract from Luo Han Guo usingwater at a temperature of 50-60° C., contacting the liquid extract withan adsorbent resin to concentrate mogrosides, eluting mogrosides withethyl alcohol, decolourising the extract with an ion exchange resin,then recovering the alcohol post decolourising and finally freezedrying.

Despite advances in processing technology, existing sweeteningcompositions derived from Luo Han Guo fruit still suffer from thedisadvantages of having a brown/yellow colour and noticeable undesirableflavours.

Thus there remains a need for sweetening compositions containing terpeneglycosides that have a clean flavour with minimal undesirable or “off”notes and and light colour. It is an object of the present invention togo at least some way towards providing such a composition or at least toprovide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a sweeteningcomposition, the composition containing from about 16% to about 75%mogroside V and from about 30% to about 95% total terpene glycosides ona dry weight basis, and wherein a filtered (0.2 μm) solution of thecomposition in water having a solids content of 1% w/v has an absorbanceat 420 nm of about 0.55 or below.

In a preferred embodiment, the composition is in the form of a powder.In another embodiment, the composition is in the form of a liquid,conveniently an aqueous solution.

Preferably, the terpene glycosides in the composition are naturallyoccurring terpene glycosides obtained from fruit of the Cucurbitaceaefamily.

Preferably the terpene glycosides in the composition are triterpeneglycosides obtained from Luo Han Guo fruit.

More preferably, substantially all of the solid components of thecomposition are obtained from Luo Han Guo fruit.

In preferred embodiments, the composition contains from about 20% toabout 70% mogroside V and from about 40% to about 90% total terpeneglycosides, such as about 30% to about 65% mogroside V and about 50% toabout 85% total terpene glycosides, such as about 35% to about 60%mogroside V and about 55% to about 85% total terpene glycosides, such asabout 40% to about 55%, mogroside V and about 60% to about 80% totalterpene glycosides, on a dry weight basis.

In preferred embodiments, the absorbance at 420 nm of a filtered (0.2μm) solution of the composition in water having a solids content of 1%w/v is less than about 0.5, more preferably less than about 0.4, such asless than about 0.35, such as less than about 0.3, such as from about0.05 to about 0.25.

In a further aspect, the present invention provides a sweeteningcomposition comprising:

-   -   (a) a first component containing from about 16% to about 75%        mogroside V and from about 30% to about 95% total terpene        glycosides on a dry weight basis, and wherein a filtered        solution (0.2 μm) of the first component in water having a        solids content of 1% w/v has an absorbance at 420 nm of about        0.55 or below; and    -   (b) one or more additional components.

The additional components may be selected from colouring agents,flavouring agents and other sweetening agents.

In preferred embodiments, substantially all of the solid components ofthe first component are derived from fruit of the Cucurbitaceae family,preferably Luo Han Guo fruit.

In a further aspect, the invention provides a beverage containing asweetening composition of the invention as described above.

In a further aspect, the invention provides a food product containing asweetening composition of the invention as described above.

In a further aspect, the invention provides a healthcare compositioncomprising a sweetening composition of the invention as described above.

In a further aspect, the invention provides a process of preparing asweetening composition containing terpene glycosides, the processcomprising the following steps:

-   -   (a) obtaining a terpene glycoside-containing liquid extract from        a fresh plant source material containing terpene glycosides;    -   (b) clarifying the extract;    -   (c) concentrating terpene glycosides in the extract to obtain a        purified terpene glycoside-containing solution;    -   (d) heating the purified terpene glycoside-containing solution        to a sufficient temperature and for a sufficient time to form        melanoidins; and    -   (e) separating melanoidins from terpene glycosides in the        solution to obtain a decolourised terpene glycoside-containing        solution.

In preferred embodiments, the process also includes the step of dryingthe decolourised terpene glycoside-containing solution obtained fromstep (e) to form a powdered composition. In preferred embodiments, thedecolourised terpene glycoside-containing solution obtained from step(e) is first concentrated before final drying.

In preferred embodiments, the fresh plant source material is a fruit ofthe Cucurbitaceae family, more preferably Luo Han Guo.

In preferred embodiments, step (a) comprises contacting macerated LuoHan Guo fruit with hot water, at a sufficient time and for a sufficienttemperature to extract triterpene glycosides from the fruit. Inparticularly preferred embodiments, the contacting is carried out usingcountercurrent extraction.

In preferred embodiments, the clarification step (b) comprisesultrafiltration of the extract.

In alternative embodiments, step (b) comprises treating the extract witha pectinase enzyme (preferably a commercial pectinase preparation) underconditions which lyse pectins and complex saccharides facilitatingclarification.

In preferred embodiments, the liquid extract obtained from step (b) isfiltered, centrifuged or decanted before step (c).

In preferred embodiments, the step (c) of concentrating terpeneglycosides comprises (i) contacting the clarified extract with anadsorbent resin, wherein the adsorbent resin binds terpene glycosides inthe extract; and (ii) eluting terpene glycosides from the resin toobtain a purified terpene glycoside-containing solution.

In preferred embodiments, the adsorbent resin used in step (c) is amacroporous polymeric adsorbent resin, such as a styrene divinylbenzenecopolymer, or divinylbenzene copolymer resin.

In preferred embodiments, step (c) is carried out in a pressurisedvessel.

In preferred embodiments, in step (c) the terpene glycosides are elutedfrom the adsorbent resin with an aqueous solution of ethanol. Inparticularly preferred embodiments, the elution is carried out using aplurality of elution steps using ethanol solutions of increasing ethanolconcentration.

In preferred embodiments, the heating step (d) comprises hearing theterpene-glycoside containing ethanol solution.(s) obtained from step(c), to both promote formation of melanoidins and evaporate ethanol,thereby allowing recovery of the ethanol.

In preferred embodiments, step (d) comprises heating the purifiedterpene glycoside-containing solution to a temperature of about 80° C.to about 120° C., for a period of time sufficient to form melanoidins.

In certain embodiments, the heating is carried out at a temperature ofabout 80° C. to about 100° C., for a time of about 120 minutes to about45 minutes.

In preferred embodiments, the decolourising step (e) comprisescontacting the terpene glycoside and melanoidin-containing solution fromstep (d) with a decolourising resin which binds the melanoidins (andoptionally other non-terpene glycoside molecules), to obtain adecolourised terpene glycoside-containing solution.

In preferred embodiments, the decolourising resin used in step (c)comprises a highly porous, macroporous, type I, strongly basic anionresin, preferably regenerated in the chloride form.

In preferred embodiments, step (e) is carried out in a pressurisedvessel.

In a further aspect, the present invention provides a process ofpreparing a sweetening composition from a fresh plant source materialcontaining terpene glycosides, characterized in that the processincludes the steps of:

-   -   (a) heating a terpene glycoside containing solution to a        sufficient temperature and for a sufficient time to form        melanoidins; where the terpene glycoside containing solution has        been obtained by a process comprising the steps of extracting        terpene glycosides from the plant source material and        subsequently clarifying the solution by removing pectin and        proteins;    -   (a) heating a terpene glycoside containing solution to a        sufficient temperature and for a sufficient time to form        melanoidins; where the terpene glycoside containing solution has        been obtained by a process comprising the steps of extracting        terpene glycosides from the plant source material and        subsequently clarifying the solution by removing pectin and        proteins;    -   (b) separating the melanoidins from the terpene glycosides in        the extract to obtain a decolourised terpene        glycoside-containing solution.

In preferred embodiments, the terpene glycoside-containing solution usedin step (a) is obtained by an extraction process comprising the steps ofcontacting macerated Luo Han Guo fruit with hot water, at a sufficienttime and for a sufficient temperature to extract triterpene glycosidesfrom the fruit, followed by clarification of the solution.

In particularly preferred embodiments, the contacting is carried outusing countercurrent extraction.

In particularly preferred embodiments, the clarification step comprisesultrafiltration of the extract.

In preferred embodiments, step (b) comprises contacting the resultingterpene glycoside- and melanoidin-containing solution with adecolourising resin that binds melanoidins (and optionally othernon-terpene glycoside molecules in the solution).

In preferred embodiments, the process includes the step of concentratingterpene glycosides in the solution. Preferably this step comprises (i)contacting the clarified extract with an adsorbent resin, wherein theadsorbent resin binds terpene glycosides in the extract; and (ii)eluting terpene glycosides from the resin to obtain a purified terpeneglycoside-containing solution.

In preferred embodiments, the adsorbent resin used in the concentrationstep is a macroporous polymeric adsorbent resin, such as a styrenedivinylbenzene copolymer, or divinylbenzene copolymer resin.

In preferred embodiments the process includes the step of drying thedecolourised terpene glycoside-containing solution obtained from step(b) to form a powdered composition.

In a further aspect, the invention provides a sweetening compositionobtained by or obtainable by a process as described above.

In a further aspect, the invention provides a food product or a beveragecontaining a sweetening composition obtained by or obtainable by aprocess as described above.

Although the invention is broadly as defined above, it is not limitedthereto and also includes embodiments of which the following descriptionprovides examples.

DETAILED DESCRIPTION OF THE INVENTION

Definition

In this specification, unless the context requires otherwise, the term“total terpene glycosides” means the total concentration of terpeneglycosides determined in a composition based on the accrual ofindividual terpene glycoside peaks observed during an HPLC analysis.Also, for the avoidance of doubt, the term “triterpene glycosides”includes without limitation both mogrosides and siamenosides.

Sweetening Compositions of the Invention

As defined above, the present invention relates to sweeteningcompositions containing terpene glycosides. The compositions arepreferably derived from fruit of the Cucurbitaceae family, preferablyLuo Han Guo fruit, containing triterpene glycosides (mogrosides andsiamenosides). The sweetening compositions of the present invention, atleast in preferred embodiments, have a cleaner flavour and lightercolour than existing sweetening compositions derived from Luo Han Guo.

In one aspect, then, the invention relates to a sweetening compositioncontaining terpene glycosides, wherein a 0.2 μm filtered solution of thecomposition in water having a solids content of 1% w/v has an absorbanceat 420 nm or about 0.55 or below. The proportions of terpene glycosidesin the sweetening compositions of the present invention may vary,depending on the natural diversification of the fruit source materialfrom which the composition is made. The compositions of the presentinvention generally contain between about 16% and about 75% mogroside Vand between about 30% and about 95% total terpene glycosides, on a dryweight basis.

In certain preferred embodiments, the compositions of the invention maycontain about 35% to about 60% mogroside V, and about 55% to about 85%total terpene glycosides on a dry weight basis.

As indicated above, the absorbance at 420 nm of a sub-micron (0.2 μm)filtered solution in water of the compositions of the present inventionhaving a solids content of 1% w/v is about 0.55 or below. The absorbanceat 420 nm correlates with the colour of the compositions—the lower theabsorbance, the lighter the colour. Also, the applicants have found thatthe absorbance at 420 nm correlates with the caramelised, cooked andmaple taste of the compositions—the lower the absorbance, the cleanerthe flavour of the compositions. The compositions of the presentinvention have an off-white to pale yellow colour, and a clean flavour,with minimal liquorice and burnt-type caramel and maple flavours thatare associated with prior art compositions.

Preferred compositions of the present invention include those in whichthe absorbance at 420 nm of a filtered (0.2 μm) solution of thecomposition in water having a solids content of 1% w/v has an absorbanceof less than about 0.3, such as about 0.05 to about 0.25.

The compositions of the present invention may be in the form of either aliquid or solid. In particularly preferred embodiments, the compositionsof the invention are in the form of a powder. In other embodiments, thecompositions may be in the form of an aqueous solution.

In certain preferred embodiments, the compositions of the inventionconsist of, or consist essentially of, material naturally occurring inand extracted from fruit of the Cucurbitaceae family, preferably Luo HanGuo fruit, ie the compositions do not include significant proportions ofsolid components from sources other than Cucurbitaceae fruit. Thesecompositions of the invention are non-nutritive, with the onlysweetening components in the compositions being terpene glycosides.

In certain alternative embodiments, the sweetening compositions of thepresent invention may be combined with other materials, such as desiredadditional flavouring, colouring and/or sweetening agents. In anotheraspect, then, the invention relates to a sweetening compositioncomprising (a) as a first component, a composition of the presentinvention as described above, containing from about 16% to about 75%mogroside V and from about 30% to about 95% total terpene glycosides ona dry weight basis, and wherein a filtered solution (0.2 μm) of thefirst component in water having a solids content of 1% w/v has anabsorbance at 420 nm of about 0.55 or below; and (b) one or moreadditional components.

In preferred embodiments, substantially all of the solid components ofthe first component are derived from fruit of the Cucurbitaceae family,preferably Luo Han Guo fruit.

The additional components may be selected from colouring agents,flavouring agents and other sweetening agents.

The compositions of the present invention may be used as sweeteningagents in various foods, beverages and confectionery products.

Preparation of Sweetening Compositions of the Invention

Melanoidins are coloured compounds formed primarily by interactionsbetween carbohydrates and compounds having a free amino group, such asfree amino acids and the free amino groups of peptides and proteins. Thecomplex network of interactions, resulting in melanoidins as the finalreaction products, are commonly referred to as the Maillard reaction.

The applicants have surprisingly found that by incorporating into aprocess of extracting terpene glycosides from a plant source materialsuch as Luo Han Guo, the step of heating a terpene glycoside-containingextract for a sufficient time and temperature to encourage the Maillardreaction and formation of melanoidins (from endogenous fruitcarbohydrate and amino-group-containing impurities remaining in theextract), the coloured melanoidin compounds formed can thereafter beremoved from the extract, conveniently using a decolourising step,furnishing a product having a clean flavour and light colour.

Processes of preparing the sweetening compositions of the presentinvention will now be described in more detail.

The sweetening compositions of the present invention can be made fromany fruit of the family Cucurbitaceae, tribe Jollifieae, subtribeThladianthinae, genus Siraitia, that contains sweet terpene glycosides.Such fruit include S. grosvenorii, S. siamensis, S. silomaradjae, S.sikkimensis, S. africana, S. borneensis and S. taiwaniana. It is howevermost preferred that the fruit used is S. grosvenorii, also referred toas Luo Han Guo fruit. The following description of the process is withreference to making sweetening compositions from Luo Han Guo fruit.

Luo Han Guo fruit is selected, stored and processed to provide a goodquality starting material preferably with a high level of sweetness.Typically the fresh fruit is then mechanically shredded or crushed. Theshredded or crushed fruit is then preferably contacted with hot water toextract mogrosides from the fruit. It is preferred that the extractionprocess includes a period immediately after the fruit is shredded orcrushed when the temperature of the water is greater than about 60° C.,preferably greater than about 80° C., sufficient to pasteurise the fruitand to inactivate endogenous enzymes (such as protease) present in thefruit. Inactivating the endogenous enzymes at this stage has thebeneficial effect of reducing enzymatic browning and limitingoff-flavour formation caused by enzymatic action. It is preferred thatthe fruit and water are well mixed to ensure even contact between thefruit and the hot water thus ensuring the enzymes are evenly exposed tothe hot water and therefore denatured as quickly as possible.

For example, a continuous countercurrent extraction process can be used,whereby the shredded fruit is fed into the countercurrent extractorwhere it is contacted with water at approximately 80° C. forapproximately 30 minutes. Countercurrent extraction processes andapparatus are known in the art. By way of example, a countercurrentextractor of the type described in U.S. Pat. No. 5,419,251 (Mantius etal), with or without the longitudinal members described, may be used inthe processes of the present invention. One advantage of using acountercurrent extraction process is that the extraction times needed toextract substantially all of the available soluble solids are typicallyless than if a conventional pot-type extraction process was used.Generally, the contact time between the fruit and the water in acountercurrent extraction process is between about 30 and 60 minuteswhereas to achieve the same extraction of soluble solids in a potprocess may take 3 separate decoctions and require contact time ofbetween 90 and 120 minutes. Another advantage is that less water isusually needed. Typically, a ratio of water to fruit of around 1.5:1will suffice in a countercurrent extraction process, whereas in apot-type multiple extraction process a water:fruit ratio of around 3:1is generally required.

Alternatively, the water may be heated to about 100° C. and the mixturedecocted for about 30 to 60 minutes, such as about 45 minutes. Thedecoction is then drained off the fruit and preferably filtered orscreened to remove large fruit pulp particles. The hot water extractionprocess may be repeated one or more times on the fruit remaining, andthe decoctions obtained from each extraction combined.

The decoction or liquid extract obtained is then preferably cooled, andclarified to provide clarity and prevent gelling of the juice. Theclarification may be carried out using any suitable method.

In preferred embodiments, the clarification is carried out by use ofultrafiltration, such as by using an ultrafiltration membrane with amolecular weight cut-off that allows for the passage of the mogrosidesin the permeate while retaining unwanted proteins and pectins in theretentate. An ultrafiltration membrane of between 50,000-100,000 daltonsis therefore preferred.

Alternatively, the clarification may be carried out by treatment withphosphoric acid or with pectinase enzyme. Pectinase enzyme may be usedconveniently in the form of a commercially available enzyme mixturecontaining pectinase enzyme, in order to lyse the pectin and precipitatepectin-stabilised peptides and protein from the liquid extract. Suitablecommercially available enzyme preparations include Novozyme 3356 andRohapect B1. Pectinase may be added as a dilute solution, in an amountof from about 0.001% to about 1%, on a dry weight basis. Convenientlythe liquid extract is held with the pectinase with gentle agitation at atemperature of about 30° C. to about 55° C., such as about 40° C. toabout 50° C., until the liquid extract is substantially free of pectin,typically for a period of about 15 to about 60 minutes, such as about 30minutes.

The liquid extract is then preferably treated to deactivate thepectinase and to denature proteins improving coagulation and theirco-precipitation with the degraded pectin. This may conveniently beachieved by heating rapidly to about 80° C. to about 90° C., such as toabout 85° C., for a time sufficient to deactivate the pectinase, such asabout 30 seconds to about 5 minutes.

Following deactivation of the pectinase plus coagulation andco-precipitation of the protein, the liquid extract is then preferablycooled sufficiently to allow it to be filtered easily to remove flockand denatured protein. The extract is typically cooled to less thanabout 65° C., more typically less than about 50° C. The filtration maybe carried out using any convenient method known in the art, such asthrough diatomaceous earth, or by cross-flow ultra- or micro-filtration.It is preferred that the extract is filtered to optical clarity (lessthan 5 NTU).

The next stage is to concentrate triterpene glycosides from theclarified liquid extract. This may conveniently be achieved bycontacting the liquid extract with an adsorbent resin that binds thetriterpene glycosides. Adsorbent resins suitable for use to harvesttriterpene glycosides include resins with a wettable hydrophobic matrixand that are suitable for contact with food, such as PVPP(polyvinylpolypyrrolidone), nylon, acrylic esters, styrenedivinylbenzene copolymers, divinylbenzene copolymers, and activatedcarbon. Such resins are commercially available. Preferred resinssuitable for use in the present invention are styrene divinylbenzenecopolymers and divinylbenzene copolymers. An example of a preferredresin is a divinylbenzene copolymer resin known as Alimentech P470 andcommercially available from Bucher-Alimentech Ltd. A preferredarrangement is to use at least 3 columns of resin operated in carouselmode. It is also preferred that this stage of the process is carried outin a pressurised vessel.

Following contacting of the liquid triterpene glycoside-containingextract with the adsorbent resin to harvest the mogrosides onto theresin, the bound triterpene glycosides are eluted from the resin using asuitable food grade solvent. For example, when the resin used isAlimentech P470, mogrosides may conveniently be eluted with successiveincrements of aqueous ethanol. By way of example, a suitable elutionsequence may involve first displacing residual liquid Luo Han Guoextract by rinsing with water, then carrying out pre-elution resinswelling using first 10% then 20% v/v ethanol in water, followed byelution of triterpene glycosides using 35%, 42%, then 50% v/v ethanol inwater and collection of the triterpene glycoside-rich mother liquor. Theresin may conveniently be rejuvenated for re-use with a 80% v/v solutionof ethanol in water followed by rinsing with water and adjustment of thepH as required. The solvent may be recovered by distillation for reuse.

The mother liquor (purified triterpene glycoside-containing solution)obtained from the adsorbent resin is subjected to a heating step, toencourage the formation of coloured melanoidins from residual proteinand peptides in the mother liquor. The preferred heating temperature andtime will depend on the residual levels of protein and peptides in themother liquor, and also the pressure at which the heating step iscarried out. The formation of melanoidins can be detected visually, asthe solution will become browner in colour.

In general, suitable temperatures for allowing the development ofmelanoidins range from about 80° C. to about 120° C. The heating timerequired will depend on the factors mentioned above, and also thetemperature at which the heating is carried out. However, preferredheating times may range from about 120 minutes to about 45 minutes, atabout 80° C. to about 100° C., such as about 120 minutes to about 90minutes at about 80° C. to about 90° C., if the heating is not carriedout at elevated pressure. It will be appreciated that if the heating iscarried out under elevated pressure, the time required for developmentof melanoidins will be reduced. For example, if the heating is carriedout at a pressure of about 1 Bar gauge and at a temperature of about120° C., a heating time of only about 10 minutes may be sufficient. Onthe other hand, if the heating is carried out at about 90° C. atatmospheric pressure, a heating time of up to about two hours may berequired.

Conveniently, as well as promoting the development of melanoidins, theheating step (d) may also serve to evaporate some of the alcohol fromthe mother liquor, allowing recovery of ethanol from the mother liquor.

After the heating step, the mother liquor is preferably cooled rapidly,preferably to about ambient temperature, conveniently to less than about30° C. The mother liquor is then subject to a decolourising step, toseparate the terpene glycosides in the solution from the melanoidins,and also preferably from other non-terpene glycoside molecules.Conveniently, this may be achieved by contacting the mother liquor witha decolourising resin that adsorbs the coloured melanoidin compounds,and other non-terpene glycoside molecules in the solution. Suitabledecolourising resins are known in the art and are commerciallyavailable. Highly porous, macroporous, type I, strongly basic anionresins, preferably regenerated in the chloride form, are examples ofresins particularly suitable for use in the present invention. Anexample of one such resin is that known as Alimentech A330, commerciallyavailable from Bucher-Alimentech Limited.

The decolourising step may be carried out in a number of stages,incorporating inter-step pH adjustment, and using a number ofchromatography columns, such as two or three columns, joined insequence, or segmented in a single column. The A330 resin may beregenerated with caustic brine followed by rinsing with hardness freewater. The mother liquor is pumped successively through the resincolumns to allow binding of melanoidins to the resin. It will also beappreciated that other non-terpene glycoside components present in themother liquor, such as proteins and flavones, will also bind to thedecolourising resin. It is preferred that the arrangement allows forinjection of a pH adjusting agent, such as dilute citric acid, into theresin columns, to maintain a pH of about 3.3 to about 4.3 in the motherliquor passing into the successive columns or column segments. It isalso preferred that the pH of the decolourised triterpeneglycoside-containing solution is then adjusted as required to about 4 orbelow, conveniently about 3.8, to inhibit colour reversion in thesubsequent concentration and drying steps.

The decolourised triterpene glycoside-containing solution is thenfurther processed to obtain a refined, bland tasting, pale colouredsweetener concentrate or a powder, as desired. The triterpeneglycoside-containing solution is preferably first concentrated beforedrying. This may conveniently be carried out by any suitable method, forexample using a spinning cone evaporator under vacuum, at a temperatureof around 65° C. The resulting concentrated solution may then be driedusing any suitable method known in the art, such as freeze-drying,vacuum-drying, spray-drying or in a forced air oven, at a temperatureapplicable to the method.

The process of the present invention may be employed on a commercialscale to make substantial quantities of sweetening compositions. It willalso be appreciated that various batches of sweetening compositions ofthe present invention containing varying levels of triterpene glycosidesmay be blended together where a consistent composition having particularproportions of mogroside V and total triterpene glycosides is desired.

The invention will now be described in further detail with reference tothe following non-limiting examples.

Examples

Examples 1, 2 and 3 below describe the preparation of three sweeteningcompositions according to the present invention, containing 54% w/wmogroside V, 39% w/w mogroside V, and 42% w/w mogroside V respectively.The percentages of mogroside V are on a dry weight basis, after dryingfor 2 hours at 85° C.

Example 1

Luo Han Guo fruit extract, (pH 4.2 and 6.2% w/w soluble solids byrefractometer), was received frozen from Guilin, P R China. The Luo HanGuo extract had been prepared by the following method.

1.2 tonnes of good quality, fresh Luo Han fruit is mechanically shreddedand dropped into a steam jacketed vat. 1,500 L of filtered water isadded to the macerated fruit. The water is heated to 100° C. and themixture is decocted for 45 minutes. After 45 minutes the decoction isdrained off the fruit and filtered to remove large fruit particles. Afurther 1,500 L of water is added to the fruit remaining in the vat. Thewater is heated to 100° C. and the mixture is decocted for 45 minutes.After 45 minutes the decoction is drained off the fruit, filtered toremove large fruit particles, and combined with the decoction from thefirst cycle. This cycle is repeated one more time to give a total volumeof 4,500 L of supernatant. The supernatant has soluble solids of between3%-6.5%.

The extract was thawed and 988 g warmed in a thermostatically controlledwater bath to 45±5° C.

1. Removal of suspended solids to avert blinding of the harvestingadsorbent resin.

A proprietary pectinase enzyme preparation (Novozym 3XL) 0.2 mL wasadded and the mixture gently stirred for 30 minutes until a flock formedand began to settle, whereupon the temperature was rapidly raised to 85°C. to terminate the enzyme activity plus held at 85° C. for anadditional 5 minutes to denature labile indigenous fruit proteins.Thereafter the separating mixture was cooled sufficiently (to <50° C.)for ease of filtration through diatomaceous earth (Celite filtercel)under vacuum, (20-30 mm Hg), yielding material, pH 4.4 and 6.1% w/wsoluble solids, Absorbance₄₂₀ 1.006, of optically bright golden brownliquid turbidity <5 NTU, (nephelometric turbidity units).

2. Concentration of mogrosides by harvesting onto Alimentech P470 US-FDAcompliant adsorbent resin.

Commercially available divinylbenzene copolymer adsorbent resin,(Alimentech P470), was prepared for contact with food as defined in USFDA 21 CFR Ch. 1, 173.65, by following the supplier's (Bucher-AlimentechLtd) instructions. 100 mL of the prepared resin was packed in a glasschromatography column (Brand Catalogue No 566 14) and the filtered LuoHan extract, previously extracted in Guislin and prepared according toSection 1, percolated through the resin at ambient temperature (21±3°C.), flow controlled at 9±1 mL/min. The mogroside depleted Luo Han juicepassing out of the column contained the fruit sugars, acids and mineralsand was set aside for separate assessment for potential use as a basefor fruit drinks.

3. Desorption, (including partial purification), of the mogrosides fromthe pregnant adsorbent resin by applying incrementing concentration of afood grade solvent.

Without disturbing the column packing, residual Luo Han juice wasdisplaced from inside the resin beads with 200 mL of potable rinsewater, flow 9±1 mL/min at ambient temperature.

All subsequent elution steps were conducted at the reduced flow rate of6±1 mL/min and at ambient temperature. Each alcohol incrementimmediately followed the previous taking care not to let the meniscusdip into the resin bed. The ethanol concentration was adjusted accordingto its specific gravity measured by hydrometer.

The adsorbent resin beads were swollen and the weakly bound lowmolecular weight fruit phenolics eluted in two steps the first being 50mL of 10% v/v ethanol, [specific gravity at 20° C. (s.g²⁰)=0.9865], then70 mL of 20% v/v ethanol, [s.g²⁰ 0.9753]. These two pre-elutionfractions were collected, the ethanol recovered for recycling and theresidual aqueous bottoms set aside for separate assessment as apotential source of “natural” antioxidants.

The mogroside was then eluted in three steps, first in 100 mL of 35% v/vethanol, [s.g²⁰ 0.9572], replaced by 100 mL of 42% v/v ethanol, [s.g²⁰0.9479] and last 100 mL of 50% v/v ethanol, [s.g²⁰ 0.9316]. The threemogroside rich eluents were combined and the ethanol recovered forrecycling using a Rotavapor (Buchi 111) under vacuum (20-30 mm Hg) at55° C. The aqueous residual material was dark brown in colour andslightly turbid, pH 3.3, soluble solids 5.4% w/w, Absorbance₄₂₀ 1.32.This mogroside rich mother liquor went forward to the heating stepdescribed in Section 4 below.

The adsorbent resin was subsequently regenerated with 100 mL of 80% v/vethanol, [s.g²⁰ 0.8605], then rinsed with water until free of alcoholthereafter permitting its repeated re-use to harvest further batches ofmogroside rich mother liquor. The ethanol was recovered for recyclingand the aqueous bottoms, which contained measurable mogroside residuals,were set aside for inclusion into subsequent production cycles addedback before the clarification step chronicled in Section 1.

4. Aeration and heat treatment to accelerate melanoidin developmentpromoting agglomeration of the protein and peptide residuals in themother liquor facilitating their subsequent adsorption by thedecolourising resin.

The mogroside rich mother liquor was transferred to an open beaker whichwas placed in a boiling water bath. Following temperature equilibrationthe mother liquor was retained at >98° C. for one hour with gentlestirring and the occasional addition of aerated water to maintain aconstant volume. Thereafter the thermally treated mother liquor wasrapidly cooled to ambient temperature. The colour of the mother liquorhad intensified and the solution was visibly turbid evidence ofmelanoidin formation. The visibility of the protein haze increased withcooling and it was readily filterable on No 4 filter paper (Whatman)providing an intensely browned filtrate, pH 3.3, soluble solids 5.3%w/w, Absorbance₄₂₀ 4.59.

5. Removal of the melanoidin colour (plus associated protein) developedin the mother liquor using a three stage decolourisation processincorporating inter-step pH adjustment, by contact with a US-FDAcompliant decolourising resin (Alimentech A330).

Commercially available ion exchange resin (Alimentech A330) was cycledrepeatedly with acidified brine (10% w/v NaCl plus 0.5% w/v HCl), water,caustic soda solution (4% w/v NaOH) and softened water in accordancewith the supplier's (Bucher-Alimentech Limited) instruction untilcompliant with US FDA 21 CFR Ch. 1, 173.25. Thereafter 20 mL aliquots ofthe compliant A330 resin were transferred to three chromatographycolumns (Brand Catalogue No 566 32) and each regenerated with 40 mL ofcaustic brine, flow controlled at 1.0±0.2 mL/min, followed by 140 mL ofdeionised rinse water at an increased flow of 4±1 mL/min. The threecolumns were then joined in sequence with an inter-stage dosing andmixing pot betwixt column 1 and 2, plus another between column 2 and 3,therein permitting the controlled injection of dilute citric acid (0.1mol/L) to maintain a pH of 3.8±0.5 in the mother liquor passingsuccessively into decolourising columns 2 and 3.

The thermally treated and filtered mother liquor was pumped sequentiallythrough the three decolourising columns, provisioned with inter-stage pHcorrection, at 2.0±0.2 mL/min followed with deionised displacement waterand the light straw coloured mother liquor collected until sweetness wasbarely perceivable to taste. The pH of the decolourised composite wasadjusted down to 3.8 with a few crystals of citric acid to obtain a palestraw coloured syrup 3.3% w/w soluble solids, Absorbance₄₂₀ 0.123. ThepH adjustment was pivotal to inhibit colour reversion in the successiveconcentration and drying steps.

6. Concentration, drying and milling to obtain a refined bland tasting,pale coloured sweetener possessing greater than 50% w/w mogroside Vcontent.

The decolourised and pH adjusted mother liquor was evaporated to 28% w/wsoluble solids in a Rotavapor (Buchi 111) under vacuum (20-30 mm Hg) at65° C. The remaining viscous amber liquid was transferred to aclock-glass and dried in a forced air oven (Clayson) at 65° C. for sixhours.

The resulting melt was allowed to solidify at ambient temperature thenhand ground with a mortar and pestle to a fine powder which was returnedto the forced air oven (65° C.) and dried to constant temperature,providing off white powder, mogroside V content 54% w/w. The pH andAbsorbance₄₂₀ of a 1.00% w/w solution reconstituted in deionised waterwas 4.0 and 0.064 respectively.

Example 2

Luo Han Guo fruit juice 4,500 L was hot water extracted from 1.2 tonnefruit using the method described in Example 1, then chill stored at 0 to−3° C. until processed. The juice was withdrawn from storage in 500 Lbatches and warmed in a steam jacketed open kettle to 45±5° C. The juicepH was 3.9 and availed 3.2% w/w soluble solids, (determinedgravimetrically).

1. Removal of suspended solids to avert blinding of the harvestingadsorbent resin.

A proprietary pectinase enzyme preparation (Novozym 33056) 100 mL wasadded to each 500 L batch and the incubated mixture gently stirred for30 minutes until a flock formed and began to settle, whereupon thetemperature was rapidly raised to 85° C. to terminate the enzymeactivity and pasteurise the juice. The liquid was then siphoned off into50 L, food-grade polyethylene carboys, capped and left to cool. Whenbelow 65° C. the juice was filtered to optical clarity by pumping itthrough a plate filter fitted with double layers of locally purchased“filter paper” overlaid with locally procured “fine grade” diatomaceousearth. The recovery for each batch varied between 420 and 460 L, of pH3.9, optically clear, moderately browned juice.

2. Concentration of mogrosides by harvesting onto US-FDA compliant,adsorbent resin.

100 L of commercially available divinylbenzene copolymer adsorbentresin, (Alimentech P470), was prepared for contact with food as definedin US FDA 21 CFR Ch. 1, 173.65, in a locally manufactured stainlesssteel chromatography column, by following the resin supplier's(Bucher-Alimentech Ltd.) instructions. Two batches of the filtered LuoHan extract, previously prepared according to Section 1, were percolatedthrough the adsorbent resin under gravity, at 55±5° C. flow vacillatingbetwixt 3 and 4 L/min dependent upon the head height. The mogrosidedepleted Luo Han juice effused from the column to waste.

3. Desorption (including partial purification) of the mogrosides fromthe pregnant adsorbent resin by applying incrementing concentration of afood grade solvent.

Without disturbing the column packing, residual Luo Han juice wasdisplaced from inside the resin beads and the column cooled with 200 Lof ambient temperature potable plant water at an increased flow of 8L/min.

All subsequent elution steps were conducted at the reduced flow rate of6±2 L/min and at ambient temperature, (35±5° C.). Each alcoholic eluentimmediately foamed the precious taking care not to let the head dip intothe resin bed. The recycled ethanol concentration was adjusted accordingto its specific gravity measured by hydrometer.

The adsorbent resin beads were swollen and the weakly bound lowmolecular weight fruit phenolics eluted in two steps the first being 50L of 10% v/v ethanol, [specific gravity at 20° C. (s.g²⁰)=0.9865], andnext 50 L of 20% v/v ethanol, [s.g²⁰ 0.9753]. These two pre-elutionfractions were collected, the ethanol recovered for recycling and theresidual aqueous bottoms discarded to waste.

The mogroside was then eluted in three steps, first in 100 L of 35% v/vethanol, [s.g²⁰ 0.9572], replenished with 100 L of 42% v/v ethanol,[s.e²⁰ 0.9479] and last 100 L of 50% v/v ethanol, [s.g²⁰ 0.9316]. Thethree mogroside rich eluents were combined and the ethanol recoveredunder vacuum, for recycling employing a locally manufactured still,initial distillation temperature 65° C. ramping to 85° C.

The adsorbent resin was subsequently regenerated with 100 L of 80% v/vethanol, [s.g²⁰ 0.8605], then rinsed with water until free of alcohol.The ethanol was distilled under vacuum for recycling and the aqueousbottoms, which contained measurable mogroside residuals, were returnedto the beginning of the ensuing batch's clarification process,chronicled in Section 1.

In an additional step the resin was further sanitized by backwashing tofluidizing the resin then washing down-flow with 200 L of QP solution(2% w/v caustic soda), thereafter rinsed with softened water, theresidual caustic neutralised with 50 L of 4% w/v citric acid solutionand finally rinsed with 500 L of potable plant water, thereby permittingthe resins repeated re-use to harvest subsequent batches of mogrosiderich mother liquor.

4. Heat treatment to accelerate melanoidin development promotingagglomeration of the protein and peptide residuals in the mother liquorfacilitating their subsequent adsorption by the decolourising resin.

Post the alcohol recovery, the temperature of the aqueous residual200±40 L remaining in the still was raised to 95±5° C. for one hour. Theresultant intensely brown and slightly turbid mother liquor was decantedinto six 50 L carboys, capped and cooled for about two hours partiallyimmersed in cold water, where after the mother liquor temperature wasdetermined to be below 50° C. and suitable for decolourisation.

5. Removal of the melanoidin colour (plus associated protein) developedin the mother liquor by contact with a US-FDA compliant decolourisingresin, employing a two stage decolourisation process incorporatinginter-stage pH adjustment.

Commercially available ion exchange resin (Alimentech A330) 25 L wastransferred to each of two identical, locally made, stainless steelchromatography columns. Therein the resin was cycled twice in accordancewith the resin supplier's (Bucher-Alimentech Limited) instructionapplying in sequence, 50 L of caustic brine (10% w/v NaCl plus 2% w/vNaOH), followed by a softened water rinse, then 50 L of citric acidsolution (4% w/v citric acid) and finally a rinse with potable plantwater, thereby ensuring compliance with US FDA 21 CFR Ch. 1, 173.25.Each column of now compliant A330 resin was regenerated with 50 L ofcaustic brine, flow controlled at 1.0±0.1 L/min, followed by 150 L ofpotable plant rinse water at an increased flow of 6±1 L/min.

The thermally treated and cooled mother liquor was transferred undergravity through one of the decolourising columns, (flow 3±1 L/min),collected and pH adjusted to 3.8±0.5, then percolated through the seconddecolourising column, (flow correspondingly 3±1 L/min). The motherliquor remaining in the columns was recovered by flushing the columns insequence with plant RO, (Reverse Osmosis), water. The effusing lightstraw coloured mother liquor was collected until sweetness was barelyperceivable to taste. The pH of the decolourised composite egressing thesecond decolourising column was adjusted down to 3.8±0.5 with citricacid crystals to obtain a pale straw coloured syrup 300±50 L. The pHadjustment was pivotal to inhibit colour reversion in the successiveconcentration and drying steps.

6. Concentration and spray drying to obtain a bland tasting, palecoloured sweetener possessing greater than 35 w/w mogroside V content.

The decolourised and pH adjusted mother liquor from each batch pair wasevaporated to 16-25% w/w soluble solids under vacuum (40-60 mm Hg) at85° C. in a single stage locally constructed evaporator. Theconcentrates from each of the 4½ batch pairs, individual volumes 3-5 L,were accumulated frozen until sufficient bulk was available for spraydrying.

Following collection of all 9 batches, the decolourised mother liquorconcentrate composite was thawed and spray dried in locally manufacturedequipment, providing off white powder, mogroside V content 39% w/w andaccrued mogrosides 61% w/w. The pH and Absorbance₄₂₀ of a 1.00% w/wsolution reconstituted in deionised water was 3.7 and 0.208respectively.

Example 3

Luo Han Guo fruit juice 4,500 L was hot water extracted from 1.2 tonnefruit using the method described in Example 1.

1. Removal of suspended solids to avert blinding of the harvestingadsorbent resin.

The juice was filtered to optical clarity by pumping it through an ultrafiltration unit with membranes having a molecular weight cut off of 100kDa. The permeate was an optically clear, pale yellow juice.

2. Concentration of mogrosides by harvesting onto US-FDA compliant,adsorbent resin.

Commercially available divinylbenzene copolymer adsorbent resin,(Alimentech P470), was prepared for contact with food as defined in USFDA 21 CFR Ch. 1, 173.65, in a locally manufactured pressurisedstainless steel column, by following the resin supplier's instructions.The filtered Luo Han extract, previously prepared according to Section1, were pumped through the adsorbent resin under pressure at less than50° C. flow vacillating between 30 and 50 L/min. The mogroside depletedLuo Han juice effused from the column to waste.

3. Desorption (including partial purification) of the mogrosides fromthe pregnant adsorbent resin by applying incrementing concentration of afood grade solvent.

Desorption of the pregnant adsorbent resin was carried out according tothe process described in example 2.

4. Heat treatment to accelerate melanoidin development promotingagglomeration of the protein and peptide residuals in the mother liquorfacilitating their subsequent adsorption by the decolourising resin.

Employing a locally manufactured still, the combined mogroside richeluents from step 3 were heated to 80±5° C. for two hours. The alcoholwas recovered and the resultant intensely brown and slightly turbidmother liquor was cooled for about one hour where after the motherliquor temperature was determined to be below 50° C. and suitable fordecolourisation.

5. Removal of the melanoidin colour (plus associated protein) developedin the mother liquor by contact with a US-FDA compliant decolourisingresin, employing a two stage decolourisation process incorporatinginter-stage pH adjustment.

Decolourising of the mother liquor was carried out according to theprocess described in example 2.

6. Concentration and spray drying to obtain a bland tasting, palecoloured sweetener possessing greater than 35% w/w mogroside V content.

The decolourised and pH adjusted mother liquor from was evaporated to30% w/w soluble solids under vacuum (40-60 mm Hg) at less than 70° C. ina single stage locally constructed evaporator.

Following evaporation, the decolourised mother liquor concentratecomposite was spray dried in locally manufactured equipment, providingoff white powder, mogroside V content 42% w/w. The Absorbance₄₂₀ of a1.00% w/w solution reconstituted in deionised water was 0.1136.

Examples 4-7

Further sweetening compositions of the present invention were preparedusing a similar method to that described in Example 1 above.

Example 8

Luo Han Guo fruit juice 142 kg was extracted from 100 kg fruit in acontinuous counter current extractor having a capacity of 10 kg per hourat a temperature of 98±2° celsius.

The juice was a light straw colour and availed 0.0% w/w soluble solids,(determined gravimetrically). This juice would be suitable as feed stockfor the process outlined in example 3.

Comparative Examples Comparative Example A

A dry sweetening composition containing mogrosides was prepared from LuoHan Guo fruit, using the method described in U.S. Pat. No. 6,124,442(Zhou et al).

Comparative Example B

A dry sweetening composition containing mogrosides was prepared from LuoHan Guo fruit, using the following method.

-   -   Fruit is crushed/broken into irregular sizes.    -   The crushed fruit is fed directly into a hopper which is then        filled with water. The water and fruit is then heated to 100        degrees.    -   Diffusion contact 40 minutes with no agitation. The liquid is        filtered and pumped into a holding tank.    -   There are two additional extractions (each with 1.5 T of water);        again the resulting liquid is filtered and stored.    -   The fourth (and final) extraction takes place, and after 40        minutes, the liquid is drained from the vessel.    -   The fruit is removed from the vessel, and the liquid from the        4^(th) extraction is returned to the vessel, and the next batch        of fruit is added.    -   The supernatant is clarified by filtering, and supernatant is        pumped to the absorber columns (D101 resin). These are 3 m tall        and 600 mm diameter. The bed depth is approx 2.5 m, giving a        bed-volume of 700 L, which is consistent with a total resin        volume of ˜3 m³.    -   Elute mogroside fraction with ethanol stepwise from 10%-95%.        Collect all eluant.    -   Distill and recover ethanol.    -   Pump eluted solution into the decolourising columns (AB-8        resin).    -   Reduce volume twenty-fold to form a concentrate.    -   Filter free of insoluble solids and spray dry at 50-150 deg C.

Analysis of Sweetening Compositions

The nitrogen contents of the sweetening compositions produced by themethods described in Comparative Examples A and B and Example 3 weredetermined using standard analytical methods: for Examples A and B:Hills labs—Catalytic Combustion (900° C., O2), separation, ThermalConductivity Detector, [Elementar VarioMAX Analyser]; for Example 3: NZLabs—AOAC 992.15, and are shown below:

Total nitrogen for Comparative Example A was 4.86%.

Total nitrogen for Comparative Example B was 3.54%.

Total nitrogen for Example 3 was 1.75%.

The sweetening compositions of Examples 1 to 6 and Comparative ExamplesA and B were analysed using high performance liquid chromatography (HPLQto determine the percentages of both individual triterpene glycosidesand total triterpene glycosides in the compositions. The HPLC methodused is described separately below. The colour of the compositions wasalso assessed by recording the absorbance at 420 nm of a 1% w/v totalsolids solution of the compositions in deionised water followingfiltration through 0.2 μm membrane. The results of the analyses are setout in Table 1 below.

TABLE 1 Analyte Comparative Comparative Example A Example B Example 5Example 6 Example 7 Example 1 Example 2 Example 3 Example 4 Colour 1.0640.848 0.510 0.129 0.189 0.064 0.208 0.1136 0.254 (Absorbance of a 1% w/vsolution recorded at 420 nm AU Mogroside V 33.2 37.2 16.1 33.1 26.2 54.139.0 42.0 38.0 % w/w¹ Σ 47.5 56.2 61.2 61.3 57.2 80.1 60.6 62.0 58.6mogrosides % w/w¹ ¹The mogroside V and other triterpene glycoside valuesare presented as a weight/weight percentage all calculated as mogrosideV equivalent in dried (85° C. for 2 hours) powder.

HPLC Procedure for Quantifying Mogroside V and Tentatively IdentifyingSeven Additional Triterpene Glycosides Found Abundant in Luo Han GuoExtract

Reference: Translation of a perfunctory Chinese document co-authored byDr Wei Ping He, (of Guilin Shili Corporation), describing in generalterms the HPLC method used to quantify mogroside V at Shili. Theprocedure specifies a Shodex Asahipak amino column and thereby isanticipated to be an adaptation of the method developed by DrTakekimatsuhar, (University of Dedoa, Japan), employed to preparepurified mogroside V, (a translation of which is not currentlyaccessible).

Standards: A purified crystalline sample of mogroside V, (Certified95.1% w/w, analysis by HPLC), isolated from Luo Han fruit, Momordicagrosvenori Swingle, was kindly donated by Guilin Bio-GFS CompanyLimited. The certified (95.1 g mogroside V/100 g), material was adoptedas the primary standard upon which all other tentatively diagnosedtriterpene glucosides were proportioned by applying an equivalent massextinction coefficient. [Triterpene glucosides, (other than mogrosideV), were tentatively identified by their photodiode ultra-violet (UV)absorbance imprint, obtained at the apex of the eluted peak, matchedagainst the UV fingerprint of the mogroside V standard].

Primary standard: Approximately 25 mg of dried (65° C. for 2 hours)crystalline mogroside V was accurately weighed using an analyticalbalance accurate to 0.1 mg.

The (23.7 mg) crystalline mogroside V was quantitatively transferred toa 50 mL volumetric flask, dissolved in 15 mL of chromatography grademethanol and made up-to 50.00 mL with 3 mM aqueous phosphoric acid. Thecorrected concentration of mogroside V in the resultant standard wasadjusted in accordance with the proportion recorded on the certificate,(e.g., 474×0.951=450 mg/L). The resulting 450 mg/L mogroside V standardwas found stable for three months when stored at ambient temperature.

Two additional standards, 50 mg/L and 200 mg/L mogroside V were preparedby serially diluting the primary standard with mobile phase “C” topermit the development of the HPLC three point calibration curve.

Detection of Mogroside V:

Triterpene glucosides possess no true chromophore so quantitationutilises the inane UV absorbance attributable to the common mogrosidecarbon backbone, herein monitored by Chanel 1 set at 205 nm with 8 nmband width. The HPLC elution stream was additionally probed at 254 nm,285 nm and 345 nm to invigilate the identification and purity estimationof the mogroside peaks, plus to provide a quasi classification of theprevalent non-mogroside, predominantly flavonoid peaks, absorbingstrongly at the nominated wavelengths.

Reported Structure of Mogroside V:

C₆₀H₁₀₂O₂₉

molecular weight 1286 g.mol⁻¹, plus two waters of crystallisation

Sample Preparation:

The Luo Han extract, or reconstituted concentrate, was brought to roomtemperature and the soluble components permitted to re-distribute fromthe pulp for ˜2 hours with occasional agitation. Those samples with highpulp content were then centrifuged to reduce the loading onto thefiltration media.

Thereafter, pulpy, or hazy extracts were filtered to optical claritythrough 0.2 μm cellulose acetate membrane syringe filter equipped with aWhatman GFB depth prefilter. The initial 5 drops of filtrate werediscarded as they can be depleted of the less hydrophilic mogrols due toadsorption onto the filter media. Approximately 6 mL of the subsequentlyfiltered sample was collected for dilution to provide a mogroside Vconcentration of between 50 and 500 mg/L in the analysed sample directlyinjected into the HPLC Any dilution was made with mobile phase “C”,(defined hereinunder), and typically was 2 to 5 fold.

The clarity of the diluted filtrate was checked immediately beforeinjection to ensure it remained optically clear as 0.2 μm filteredcommercial Luo Han extract contains sufficient residual oligomericpectates to sporadically re-haze in the dilution solvent. When necessarythe injected sample was again filtered through a 4 mm diameterdisposable solvent resistant (PTFE) membrane. (Extracts that aredifficult to filter my require addition of filter aids. Before applyinga commercial acid pectinase enzyme to reduce the viscosity anddestabilise the pulp, ensure the elected enzyme preparation containsnegligible side activity capable of hydrolysing glucose moieties,thereby altering the relative proportions of triterpene glycosidesdetected).

Instrumentation:

All analyses were completed on a Shimadzu Class VP liquid chromatographyunit with quaternary gradient capability and a photodiode array D₂detector. The defining parameters specific to the Shimadzu apparatus arecollated hereinunder.

Chromatographic Conditions for Binary Gradient Separation:

-   -   Solvent A: Acetonitrile 1% by volume in water, for purging the        HPLC apparatus.    -   Solvent B: Acetonitrile 5% by volume in aqueous phosphoric acid,        3 mMol in total solvent.    -   Solvent C: Acetonitrile 40% by volume in aqueous phosphoric        acid, 3 mMol in total solvent.    -   Solvent D: Methanol 100% for wetting the reverse phase column        and flushing to restoring its theoretical plate count.

All the aqueous solvents were pre-filtered through a disposable, 0.2 μmcellulose hydrophilic membrane, syringe filter, before addition to thebulk solvent carboys feeding the HPLC.

-   -   Method: Mogroside V.met    -   Pump: 1.0 mL/min, sum of binary gradient components.    -   Mixer: Static low pressure mixer with 0.5 mL hold-up volume.    -   Injection loop: Rheodyne 20 μL    -   Guard column: Re-packable, Adsorbosphere C18, 5 μm, 10 mm.    -   Column: Allsphere Hexyl 5 μm, C6, 4.6×250 mm.    -   Column oven: 35° C.    -   Run time: 62.05 minutes    -   Average Retention Time and Response Factors Applied:

Retention time Window Compound (min) (min) Response factor Phenolic A24.2 2.4 3.48996 × 10⁻⁵ Phenolic B 25.5 2.5 3.48996 × 10⁻⁵ Phenolic C26.3 2.6 3.48996 × 10⁻⁵ Triterpene glycoside I 32.4 3.2 2.16732 × 10⁻⁴Mogroside V 33.3 3.3 ¹2.16732 × 10⁻⁴   Triterpene glycoside 34.8 3.52.16732 × 10⁻⁴ II Triterpene glycoside 35.4 3.5 2.16732 × 10⁻⁴ IIITriterpene glycoside 36.2 3.6 2.16732 × 10⁻⁴ IV Triterpene glycoside V41.1 4.1 2.16732 × 10⁻⁴ Triterpene glycoside 43.7 4.3 2.16732 × 10⁻⁴ VITriterpene glycoside 44.1 4.4 2.16732 × 10⁻⁴ VII ¹This response factoris the average response from the three point calibration curve developedusing the provided 95.1% certified mogroside V standard. The remainderof the triterpene glycoside response factors have been assigned on thepremise of their exhibiting an identical mass extinction coefficient tomogroside V. The total mogrosides shown in Table 1 were also calculatedusing this same assumption

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thescope and spirit of the invention. It will be readily apparent to oneskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention. The invention illustratively describedherein suitably may be practised in the absence of any element orelements, or limitation or limitations, which is not specificallydisclosed herein as essential. Thus, for example, in each instanceherein, in embodiments or examples of the present invention, the terms“comprising”, “including”, “containing” etc are to be read expansivelyand without

1. A sweetening composition, the composition containing from about 16%to about 75% mogroside V and from about 30% to about 95% total terpeneglycosides on a dry weight basis, and wherein a filtered (0.2 μm)solution of the composition in water having a solids content of 1% w/vhas an absorbance at 420 nm of about 0.55 or below.
 2. A compositionaccording to claim 1, wherein the terpene glycosides in the compositionare naturally occurring terpene glycosides obtained from fruit of theCucurbitaceae family.
 3. A composition according to claim 1, wherein theterpene glycosides in the composition are triterpene glycosides obtainedfrom Luo Han Guo fruit.
 4. A composition according to claim 1, whereinsubstantially all of the solid components of the composition areobtained from Luo Han Guo fruit.
 5. A composition according to claim 1,wherein the composition contains from about 20% to about 70% mogroside Vand from about 40% to about 90% total terpene glycosides, on a dryweight basis.
 6. A composition according to claim 1, wherein thecomposition contains about 30% to about 65% mogroside V and about 50% toabout 85% total terpene glycosides, on a dry weight basis.
 7. Acomposition according to claim 1, wherein the composition contains about35% to about 60% mogroside V and about 55% to about 85% total terpeneglycosides, on a dry weight basis.
 8. A composition according to claim1, wherein the composition contains about 40% to about 55% mogroside Vand about 60% to about 80% total terpene glycosides, on a dry weightbasis.
 9. A composition according to claim 1, wherein the absorbance at420 nm of a filtered (0.2 μm) solution of the composition in waterhaving a solids content of 1% w/v is less than about 0.5.
 10. Acomposition according to claim 1, wherein the absorbance at 420 nm of afiltered (0.2 μm) solution of the composition in water having a solidscontent of 1% w/v is less than about 0.4.
 11. A composition according toclaim 1, wherein the absorbance at 420 nm of a filtered (0.2 μm)solution of the composition in water having a solids content of 1% w/vis less than about 0.35.
 12. A composition according to claim 1, whereinthe absorbance at 420 nm of a filtered (0.2 μm) solution of thecomposition in water having a solids content of 1% w/v is less thanabout 0.3.
 13. A composition according to claim 1, wherein theabsorbance at 420 nm of a filtered (0.2 μm) solution of the compositionin water having a solids content of 1% w/v is from about 0.05 to about0.25.
 14. A composition according to claim 1, wherein the composition isa powder.
 15. A composition according to claim 1, wherein thecomposition is a liquid.
 16. A composition according to claim 15,wherein the composition is an aqueous solution.
 17. A sweeteningcomposition comprising: (a) a first component containing from about 16%to about 75% mogroside V and from about 30% to about 95% total terpeneglycosides on a dry weight basis, and wherein a filtered solution (0.2μm) of the first component in water having a solids content of 1% w/vhas an absorbance at 420 nm of about 0.55 or below; and (b) one or moreadditional components.
 18. A sweetening composition according to claim17, wherein the additional components are selected from the groupconsisting of colouring agents, flavouring agents and other sweeteningagents.
 19. A composition according to claim 17, wherein substantiallyall of the solid components of the first component are derived fromfruit of the Cucurbitaceae family, preferably Luo Han Guo fruit.
 20. Abeverage containing a sweetening composition according to claim
 1. 21. Afood product containing a sweetening composition according to claim 1.22. A health care composition comprising a sweetening compositionaccording to claim
 1. 23. A process of preparing a sweeteningcomposition containing terpene glycosides, the process comprising thefollowing steps: (a) obtaining a terpene glycoside-containing liquidextract from a fresh plant source material containing terpeneglycosides; (b) clarifying the extract; (c) concentrating terpeneglycosides in the extract to obtain a purified terpeneglycoside-containing solution; (d) heating the purified terpeneglycoside-containing solution to a sufficient temperature and for asufficient time to form melanoidins; and (e) separating melanoidins fromterpene glycosides in the solution to obtain a decolourised terpeneglycoside-containing solution.
 24. A process according to claim 23,including the step of drying the decolourised terpeneglycoside-containing solution obtained from step (e) to form a powderedcomposition.
 25. A process according to claim 24, wherein thedecolourised terpene glycoside-containing solution obtained from step(e) is concentrated before final drying.
 26. A process according toclaim 23, wherein the fresh plant source material is a fruit of theCucurbitaceae family.
 27. A process according to claim 23, wherein thefresh plant source material is Luo Han Guo fruit.
 28. A processaccording to claim 23, wherein step (a) comprises contacting maceratedLuo Han Guo fruit with hot water, at a sufficient time and for asufficient temperature to extract triterpene glycosides from the fruit.29. A process according to claim 28, wherein the extraction is carriedout using counter current extraction.
 30. A process according to claim23, wherein the clarification step (b) comprises ultrafiltration of theextract.
 31. A process according to claim 23, wherein the liquid extractobtained from step (b) is filtered, centrifuged or decanted before step(c).
 32. A process according to claim 23, wherein the step (c) ofconcentrating terpene glycosides comprises (i) contacting the clarifiedextract with an adsorbent resin, wherein the adsorbent resin bindsterpene glycosides in the extract; and (ii) eluting terpene glycosidesfrom the resin to obtain a purified terpene glycoside-containingsolution.
 33. A process according to claim 32, wherein the adsorbentresin used in step (c) is a macroporous polymeric adsorbent resin.
 34. Aprocess according to claim 32, wherein the adsorbent resin used in step(c) is a styrene divinylbenzene copolymer or divinylbenzene copolymerresin.
 35. A process according to claim 32, wherein step (c) is carriedout in a pressurised vessel.
 36. A process according to claim 32,wherein the terpene glycosides are eluted from the adsorbent resin withan aqueous solution of ethanol.
 37. A process according to claim 36,wherein the elution comprises a plurality of elution steps using ethanolsolutions of increasing ethanol concentration.
 38. A process accordingto claim 36, wherein the heating step (d) comprises heating theterpene-glycoside containing ethanol solution(s) obtained from step (c),to both promote formation of melanoidins and evaporate ethanol, therebyallowing recovery of the ethanol.
 39. A process according to claim 23,wherein step (d) comprises heating the purified terpeneglycoside-containing solution to a temperature of about 80° C. to about120° C., for a period of time sufficient to form melanoidins.
 40. Aprocess according to claim 39, wherein the heating is carried out at atemperature of about 80° C. to about 100° C., for a period of time ofabout 120 minutes to about 45 minutes.
 41. A process according to claim23, wherein the decolourising step (e) comprises contacting the terpeneglycoside and melanoidin-containing solution from step (d) with adecolourising resin which binds the melanoidins, to obtain adecolourised terpene glycoside-containing solution.
 42. A processaccording to claim 41, wherein the decolourising resin used in step (e)comprises a highly porous, macroporous, type I, strongly basic anionresin.
 43. A process according to claim 41, wherein step (e) is carriedout in a pressurised vessel.
 44. A process of preparing a sweeteningcomposition from a fresh plant source material containing terpeneglycosides, characterized in that the process includes the steps of: (a)heating a terpene glycoside-containing sohidon to a sufficienttemperature and for a sufficient time to form melanoidins; wherein theterpene glycoside-containing solution has been obtained by a processcomprising the steps of extracting terpene glycosides from the plantsource material and subsequently clarifying the solution by removingpectin and proteins; (b) separating the melanoidins from the terpeneglycosides in the extract to obtain a decolourised terpeneglycoside-containing solution.
 45. A process according to claim 44,wherein the terpene glycoside-containing solution used in step (a) isobtained by an extraction process comprising the steps of contactingmaccrated Luo Han Guo fruit with hot water, at a sufficient time and fora sufficient temperature to extract triterpene glycosides from thefruit.
 46. A process according to claim 45, wherein the contacting iscarried out using countercurrent extraction.
 47. A process according toclaim 45, wherein the clarification step comprises ultrafiltration ofthe extract.
 48. A process according to claim 45, wherein step (b)comprises contacting the resulting terpene glycoside- andmelanoidin-containing solution with a decolourising resin that bindsmelanoidins (and optionally other non-terpene glycoside molecules in thesolution).
 49. A process according to claim 45, including the step ofconcentrating terpene glycosides in the solution.
 50. A processaccording to claim 49, wherein the concentrating step comprises (i)contacting the clarified extract with an adsorbent resin, wherein theadsorbent resin binds terpene glycosides in the extract; and (ii)eluting terpene glycosides from the resin to obtain a purified terpeneglycoside-containing solution.
 51. A process according to claim 50,wherein the adsorbent resin used in the concentration step is amacroporous polymeric adsorbent resin, such as a styrene divinylbenzenecopolymer, or divinylbenzene copolymer resin.
 52. A process according toclaim 45, including the step of drying the decolourised terpeneglycoside-containing solution obtained from step (b) to form a powderedcomposition.
 53. A sweetening composition obtained by a processaccording to claim
 23. 54. A food product or a beverage containing asweetening composition obtained by a process according to claim 23.